Instruments designed to gather precise fluorescence intensity data are commonly referred to as fluorometers (also known as fluorimeters). The fluorometers found in high performance liquid chromatography (HPLC), capillary electrophoresis (CE), and automated DNA sequencing instruments are also referred to simply as fluorescence detectors. Conceptually similar fluorescence detectors are employed in microwell plate readers and microarray scanners. Other quantitative analysis applications of fluorometers include counting cells via flow cytometry, determining the amount of DNA or RNA in a sample, measuring enzyme activity, and determining concentrations of hydrocarbons or chlorophyll in water.
Fluorometric apparatuses can be differentiated by the nature of the sample, how the sample is presented to the fluorometer, and the type of fluorescence data that is gathered. In order to fully comprehend our invention and its significance, one must recognize and understand the strengths and weaknesses of the many known variations of fluorometers. At a minimum, every fluorometer incorporates an excitation light source that serves to induce fluorescence in the sample, a means to isolate only those fluorescence photons with a specified wavelength range, and a photodetector that converts the fluorescence light flux within the selected wavelength range to an analog electrical signal; many fluorometers have provision for converting the analog electrical signal to a digitized representation that can be read visually or stored for subsequent data analysis.
The process of fluorescence is initiated when molecules in the sample absorb photons from the light source. The energy that was carried by the excitation photons is transferred to the molecules, thereby creating a population of electronically excited molecules. The molecules cannot remain in these excited states indefinitely owing to several possible de-excitation pathways, one of which is photon emission (fluorescence). Owing to certain vibrational relaxation and internal conversion processes that occur between the act of photon absorption (excitation) and photon emission (fluorescence), the average wavelength of the emitted photons is invariably longer than the excitation wavelength that was used to create the excited states via photoabsorption. Within a few picoseconds of the time an excited state molecule is created, it relaxes to the first excited singlet state and it is from this state that the fluorescence occurs. The average residence time of the molecule in the first excited singlet state in usually on the order of 0.1–100 nanoseconds. The shape of the fluorescence spectrum (but not the total intensity) for any particular compound is nearly the same regardless of the choice of excitation wavelength. Likewise, the shape of the excitation spectrum (but not the total intensity) of any particular compound is nearly the same regardless of the choice of wavelength at which the emission is monitored.
Many different excitation sources can supply the more or less monochromatic incident beam of light that is needed to excite (induce) fluorescence in the sample. Some excitation light sources, including tungsten or quartz-halogen lamps, xenon arc lamps, and xenon flashlamps, emit photons over such a broad range of wavelengths so as to require that an interference filter, monochromator, or other wavelength-selector be interposed between the excitation light source and the sample. The primary purpose of the excitation wavelength-selector is to prevent scattered excitation photons whose wavelength is the same as the fluorescence signal of interest from entering the detection system. The output of medium or high pressure xenon arc lamps and xenon flashlamps covers from the vacuum ultraviolet (wavelengths shorter than 200 nm) through the ultraviolet and visible regions and into the near-infrared; thus, essentially any desired wavelength can be obtained by appropriate choice of the excitation wavelength selector, albeit at the price of having to discard 99% or more of the photons emerging from the excitation light source. Light emitting diodes (LEDs) provide photons in comparatively narrower wavelength ranges, 50–100 nm, which eases the task for wavelength filtering their output. Inexpensive LEDs that span the wavelength range from approximately 360 nm into the near-infrared are commercially available.
Laser excitation sources can be highly advantageous for fluorometer applications because their output is so highly monochromatic and the laser light can easily be directed to and focused on the desired sample location. The laser sources that are found in nearly all automated DNA analyzers and most microarray readers generally provide photons in a single, very narrow wavelength range. In order to retain at least a portion of the valuable information that is inherent in the dependence of the fluorescence intensity on excitation wavelength, such instruments may incorporate several fixed wavelength laser sources, although this increases complexity, cost, and measurement time. Tunable lasers or optical parametric oscillators (OPOs) are coherent sources whose output wavelength is continuously variable, but they are also generally large and expensive.
The fluorescence intensity can be monitored within a single emission wavelength range, at several discrete emission wavelengths, or over a continuous range of wavelengths. Instruments that employ dielectric interference filters or glass cut-off filters to select the emission monitoring wavelengths are generally referred to as fluorometers or fluorimeters. The operator may be required to select and install a different filter in the instrument every time the wavelength at which the emission is monitored is changed. Versions with several filters installed in a rotatable filter wheel or on a filter slide, which could be either manually controlled or attached to a motor, are more convenient. Monochromators are very flexible and versatile instruments for wavelength selection. Adjusting the position of a grating or prism within the monochromator allows continuous variation of the passband wavelength. The width of the passband is similarly adjustable through control of the entrance and exit slit widths. Fluorescence measurement instruments that incorporate scanning monochromators for continuous variation of the emission wavelength or both the excitation and emission wavelength are generally referred to as spectrofluorometers or spectrofluorimeters. Yet another option is to use an array detector such as a charge-coupled device (CCD) camera to collect the entire fluorescence spectrum at once. In this case, the monochromator that is used to disperse (spatially separate) the fluorescence is commonly referred to as a spectrograph. Well-known procedures can be applied to correct the experimental emission spectrum and the excitation spectrum for the wavelength dependence of the measurement system. The corrected spectra then represent fundamental fluorescence properties of the molecules, although these properties may exhibit some dependence on the molecular environment; e.g., the fluorescence spectrum could shift in wavelength if the polarity of the solvent is varied. The practice and principles of fluorescence spectroscopy are described in many textbooks and reference books.
Fluorescence lifetime is another molecular property that is less affected by details of the measurement system than is the case for the spectra. For example, the fluorescence lifetime does not change when the amount of light directed onto the sample is reduced with a neutral density filter, after a change in excitation wavelength, or if the pulse repetition frequency of the light source is varied. The individual excited state persistence times for a population of identically prepared molecules is statistically distributed, but the decay of the collective excited state population follows so-called first order kinetics or exponential decay. The lifetime is the time interval over which the excited state population falls to 1/e=36.8% of its initial population. The excited state lifetime is related to the rate constants for all process that deactivate the excited state, but it is commonly referred to as the fluorescence lifetime because fluorescence is by far the most convenient way to follow the changes in excited state population.
Little or no fluorescence lifetime information can be gained if the intensity of the excitation beam directed on to the sample is essentially constant. One means of obtaining lifetime information is to temporally modulate the intensity of the excitation light, usually in a sinusoidal pattern. The emission response of the sample necessarily has the same modulation frequency as the excitation. However, the inherent time lag between the excitation and emission processes induces a phase shift that is mathematically related to the fluorescence lifetime. Such techniques are commonly referred to as frequency domain spectroscopy.
A conceptually simpler approach is to excite the fluorescence with a light pulse of short duration and to measure the temporal pattern of the subsequent fluorescence. The entire fluorescence decay curve can be measured following a single laser excitation pulse with a digital oscilloscope or transient digitizer, whose function is to track the output of a photomultiplier tube or other photodetector at closely-spaced time intervals. A plot of fluorescence intensity vs. time interval expressed relative to the time at which the excited state population is generated is commonly referred to as a fluorescence decay curve; a digitized representation of a transient signal as a function of time is also commonly referred to as a waveform or profile. In the ideal case that the time duration (pulse width) of the excitation pulse is much shorter than the fluorescence decay time, the lifetime can be determined from a plot of In It vs. t where It is fluorescence intensity at-time t relative to the laser pulse. Many mathematical deconvolution techniques are available for situations in which the excitation pulse duration is not infinitesimally short compared to the fluorescence lifetime. Deconvolution techniques require that the intensity be measured as a function of time for both the excitation pulse and the subsequent fluorescence pulse. Apart from a relatively uninteresting multiplicative factor, the mathematical relationship between the fluorescence and excitation waveforms involves a single parameter, namely the fluorescence lifetime. Each deconvolution procedure has the same goal, namely to determine the value of the lifetime that gives the best fit between the observed and predicted fluorescence decay curves.
The statement above that the fluorescence lifetime is independent of the emission monitoring wavelength is not necessarily true for mixtures. The apparent fluorescence lifetime will depend on the excitation or fluorescence wavelength if the sample contains multiple emitting species with different lifetimes and different excitation and emission spectra. In such cases, one expects to observe bi-exponential or multi-exponential decay. The invariance of the fluorescence lifetime to excitation or emission wavelength is a test of sample purity, similar to tests based on the invariance of the excitation spectrum to emission monitoring wavelength and the invariance of the emission spectrum to excitation wavelength. The mathematical data processing techniques, including deconvolution, are readily generalized to account for multiple emitting species.
The traditional way to gather the fluorescence decay curve (and the laser excitation pulse shape, if needed for deconvolution) is via time-correlated single photon counting (TCSPC). In TCSPC the sample is repetitively excited and a histogram of the time interval between when the sample is excited and when the first fluorescence photon is detected is generated. The histogram is functionally equivalent to the fluorescence decay curve that can be measured with a transient digitizer. The data contained within the TC-SPC histogram follow so-called Poisson statistics. On the other hand, in order to attain the condition of Poisson statistics, the measurement conditions must be arranged so that an actual datum (one point in the histogram) is collected on no more than 1 or 2 percent of the laser pulses. Thus, data collection is a lengthy and inefficient process.
Fluorometry often provides higher measurement sensitivity and specificity, greater ease of operation, faster measurement time, or lower instrumentation cost in comparison to other instrumental techniques. Fluorescence spectroscopy is inherently sensitive because the signals of interest are measured against a low (ideally zero) background signal. Absorption spectroscopy, in contrast, is less sensitive when operating near the limit of detection or limit of quantitation because a very small decrease in a large light signal must be determined. The unique combination of excitation spectrum, emission spectrum, and lifetime possessed by each fluorescent compound provides the specificity.
The fluorescent signal intensity depends, inter alia, on the flux of excitation photons within the sample volume and the number of fluorophores within that volume. Other factors that influence the total fluorescence intensity are the wavelength-dependent responses of the wavelength analyzer and the photodetector, the optics used to deliver the excitation light to the sample, the optics used to deliver a portion of the emitted light to the wavelength analyzer in front of the photodetector; and the specific geometrical arrangement of the light source, excitation optics, collection optics, and wavelength analyzer. The fluorescence intensity thus depends on inherent spectroscopic properties of the potentially fluorescent molecules (fluorophores), on the concentration of fluorophores, and on properties of the measurement system itself.
The procedures for characterizing the measurement system properties are tedious and time consuming. Therefore, for purposes of quantitative analysis one generally compares the fluorescence intensity of the sample to the fluorescence intensities of reference or standard samples whose concentrations are known. If the sample consists of a fluid solution, the concentration is usually expressed as a mass per unit volume. For fluorescent species arrayed on a surface, the amount would likely be expressed in terms of mass per unit area. Therefore, fluorescence induced in a sample makes it possible to identify if a fluorescent compound is present in a sample (qualitative analysis) and, if so, to determine its concentration or amount (quantitative analysis).
If it is known that the sample fluorescence intensity arises from a single, known compound, implementation of the quantitative analysis techniques and interpretation of the data are straightforward. The quality and value of the analysis is compromised if the sample contains unknown or unsuspected fluorescent species or if the fluorescence data are corrupted by interfering background signals. Fluorescence is ideally a zero background technique, as was stated above, but a certain amount of background signal is inevitably present. The sources of the background signal are many, including stray excitation light at the desired fluorescence monitoring wavelength, fluorescence from impurities in the sample, and interfering fluorescence of the sample container.
A high data acquisition rate is essential for most chromatographic analyses, microplate or microarray scanning, in vivo optical diagnostics, and many other procedures in which either the sample composition is rapidly changing or many different samples must be tested. How to account for background signal and how to sense when more than one species is contributing to the fluorescence signal is a common theme and challenge. Confirmatory chemical analysis by techniques that rely on discrete sampling are so time consuming as to be completely incompatible with the desire for rapid measurement rate.
A primitive approach that has some value for chromatography is to examine the pattern of intensities at contiguous elution times. The fluorescence intensity of a species as it elutes is expected to vary smoothly from zero to a maximum and then return to zero. Various mathematical formulas have been postulated to fit the shapes of the peaks, which are referred to by such terms as normal (Gaussian) or log-normal; sufficiently large deviations from the characteristic shape for compounds eluting at comparable time intervals after the sample was injected could signify the presence of two or more fluorophores whose peaks are overlapping. As long as the sample concentrations are low enough so that energy transfer and quenching processes are negligible, the total fluorescence intensity is closely approximated by the sum of contributions from the individual fluorescent compounds in the sample. The sample conditions that apply to high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), for DNA sequencing analysis, and for many other fluorescence procedures satisfy the dilute sample condition requirement. Thus, one can attempt to resolve the overlapping peaks, but procedures that attempt to do so solely on the basis of lineshape are notoriously inaccurate. Nor does such an analysis provide any information on the chemical identity of an interfering fluorophore. Background subtraction techniques that assume that the background signal is either constant or slowly varying are similarly applied and have similar limitations.
There is precedent for using spectroscopic data in more elaborate fashion to test for peak purity. For example, photodiode array (PDA) detectors that can measure a full absorption spectrum, as opposed to absorbance at a single wavelength, are well known in chromatography. Peaks can be tentatively assigned and peak purity assessed by comparing the measured spectrum at a given elution time to the entries in a database of known standard spectra. A peak purity index is derived from the degree of overlap of the unknown spectrum with its closest match in the database. However, if the peak purity index is low, suggesting that there is more than one emitting component in the sample, the problem of how to apportion the total spectrum into its components, including background signal, remains. Thus, PDA detectors are used more to avoid misassignments than it is to increase the amount of information that can be gained in a given amount of experiment time.
Owing to the cumbersome nature of the peak purity testing procedures and the lack of easily applied algorithms that can accurately resolve overlapping peaks into the contributions of individual species, great effort is undertaken to arrange the chromatographic separation conditions to reduce the likelihood that more than one kind of species is in the detector volume at a given time. Unfortunately, these conditions, which require careful optimization and adjustment of variables such as the solvent's eluting strength and the flow rate, invariably result in much longer elution times and diminished productivity.
In fact, virtually all fluorescence detectors used in chromatography, microplate readers, microarray readers, quantitative PCR apparatuses, etc., rely on measuring with a single excitation wavelength and a single emission wavelength for each sample composition or location because this is the only approach compatible with the high data acquisition rates. One must recognize that the datum from such a measurement is simply a number, regardless of the units in which it is expressed, e.g., current, voltage, counts, etc. The data are dimensionally zero-order in mathematical terms. It should be apparent that unambiguously decomposing this number into the separate contributions of different fluorophores or a fluorophore and background is impossible. From the standpoint of purity, it is similarly impossible mathematically to assign a purity index to the individual measurement.
The only fluorescence detectors that routinely collect a full fluorescence spectrum at closely spaced time intervals, e.g., less than one second, are found in very expensive automated DNA sequencers. The most sophisticated of these sequencers collect the entire fluorescence spectrum with a CCD camera positioned at the exit focal plane of a spectrograph, but most of the spectral information is discarded in the data processing step. Other versions make measurements at a multiplicity of wavelengths (typically four because four dyes are used in one-lane DNA sequencing) via rapid rotation of a filter wheel or the use of dichroic filters to direct the light in various wavelength ranges to multiple detectors. Certain microplate and microarray readers allow either the emission monochromator or excitation monochromator to be scanned to generate a full spectrum, but these modes are too slow for most applications.
Fluorescence potentially offers many different options (none of which are routinely used) for confidence testing analogous to the use of a PDA in absorbance detection for HPLC. The analogy would be closest if a complete fluorescence spectrum were measured at each elution time in the chromatogram, which could be accomplished with an intensified photodiode array (IPDA), also referred to as a gated optical multichannel analyzer (OMA). Alternatively, a CCD camera detector with elements binned along an axis perpendicular to the spectral dispersion direction could be used to collect a full fluorescence spectrum. Although such implementations have been described in the literature, their use has been limited to research purposes because of high cost and other reasons.
There is ample evidence in the literature and widespread agreement among researchers that multidimensional fluorescence analyses yield much more information in terms of both specificity and sensitivity than corresponding one-dimensional spectral techniques. However, the use of multidimensional techniques has largely been limited to research investigations because: 1) The rate at which the data are gathered and processed is generally far too slow for any practical commercial application; 2) Technologies that could achieve the requisite speed are prohibitively expensive; and 3) Robust and rapid data analysis methodologies are not available to utilize the information that is inherently contained in the data. Attempts at commercialization of the technology and methodology have been hampered by these impediments.
Fluorescence is unique among spectroscopic techniques in its capability for multidimensional data wherein fluorescence intensity data are measured along at least two of the three important spectroscopic coordinates, which are excitation wavelength, emission wavelength, and fluorescence decay time. The most familiar multi-dimensional fluorescence representation is that of an excitation-emission matrix (EEM). EEMs are most commonly generated as a series of emission spectra acquired at different excitation wavelengths. Alternatively and equivalently, a series of excitation spectra can be gathered for different emission monitoring wavelengths and will yield the same result. By their very nature, EEMs contain more information than is available in either the excitation or the emission spectrum alone. The potential benefits of EEMs for purposes of diagnosing tumors via endoscopy or identifying sources of oil spills have long been recognized. However, the practical use of EEMs has been severely circumscribed by the lengthy and tedious manner in which they must be acquired.
At least two groups have proposed speeding the process by which EEMs are collected using a multiple wavelength excitation source based on Raman shifting, but these are complicated instruments requiring separate pairs of optical fibers for every excitation wavelength and an expensive CCD camera. Moreover, the Raman shifting process leads to large fluctuations in the laser excitation pulse energy and degraded signal to noise. A company has recently introduced a commercial fluorimeter that incorporates an old technique known as video fluorometry, allowing the collection of an EEM in as short a time as one second. However, the fast measurement time comes at a ten-fold or greater sacrifice in measurement sensitivity and the question of how to analyze the data remains.
Decomposing the sample's total emission or excitation spectrum into contributions from its various constituents is difficult. If a pulsed excitation source of sufficiently short duration is employed, one can collect second-order data in the form of a wavelength-time matrix (WTM). A WTM in its simplest incarnation consists of fluorescence decay curves measured at a series of emission or excitation wavelengths. The information can be assembled into a two-dimensional data array in which the columns represent different wavelengths (either excitation or emission), and the rows represent different time increments relative to the time at which fluorescence was excited with a short duration laser pulse. Although WTMs have received far less attention in the literature than EEMs, they possess certain advantages owing to the manner in which the fluorescence decay curves can be mathematically related to the laser excitation waveforms.
If EEMs or WTMs are collected in sequence mode, i.e., one emission spectrum or one fluorescence decay curve at a time, it is very important that conditions be held as constant as possible during the entire sequence to avoid distortion. The shorter the measurement time for a fluorescence decay curve, the easier it is to approach the case of constant sample conditions. Two likely sources of distortion are drifts in the laser power or sample degradation. For example, if the laser intensity steadily dropped during the collection of the EEM, then there will be a systematic error across the EEM. The same type of behavior results if photochemistry or other processes change the concentration of fluorophores in the sample during the course of the data collection. These problems are avoided if the entire EEM or WTM can be collected simultaneously.
Heretofore, instruments used for generating WTMs have been too slow and unstable to be useful for many analytical processes, such as analysis of samples whose properties change rapidly in time and space, including analysis of flowing fluids or rapidly scanning sample surfaces. The reasons for this situation are many and varied, but include shot-to-shot laser fluctuation, slow repetition rates and expense of the lasers, inability of digitizers to keep pace with lasers having faster repetition rates, lack of methodology for handling the volume of data generated, and lack of robust algorithms for analysis of the data.
Our invention solves numerous problems related to the pervasive and challenging situation in which the sample contains multiple fluorescent compounds.